The present invention relates to a method of and means for the recovery of substantially pure messenger ribonucleic acid (hereinafter referred to as mRNA) fraction from a mixture of a plurality of such fractions. Such purification is required for various molecular biological tests, as well as for the purposes of gene therapy and genertic engineering. Hitherto no satisfactory method for the preparative recovery of essentially pure mRNA fractions is known and it is the object of the present invention to provide such a method.
mRNA is a type of ribonucleic acid (RNA) that is present in the biological and cells is recoverable therefrom, amounting to about 2-3% by weight of the total RNA. There are known methods for the separation of the group of mRNA from the remaining RNAs isolated from a particular cell, but problems have been encountered in the preparative resolution of the group of mRNAs into its constituent component.
According to a known method for the separation of the group of mRNA fractions contained in the total RNA recovered from a cell, the total RNA is chromatographed on a column of powderous cellulose or powderous agarose bearing polyuridylic acid (hereinafter "poly-U") or polythymidylic acid (hereinafter "poly-dT") residues. in this manner, the group of mRNA fractions contained in a cell is separated from all the remaining RNAs. The method is based on the reversible binding of mRNA to the poly-U or poly-dT residues via H-bridges.
It is also known to resolve the group of mRNA fractions into its constituent mRNA fractions by means of electrophoresis, using, for example, an agarose or polyacrylamide gel as matrix. Upon the completion of the electrophoresis each constituent mRNA occupies a different zone on the matrix. There have also been reported various transfer techniques by which various biological macromolecules can be transferred to a receiver carrier such as a blotting paper (see for example Southern, (1975) J. Mol. Biol. 98 503-517; Bittner et al. (1980) Analytical Biochemistry 102 459-471; PeFeroen et al. (1982) Febs. Lett. 145 369-372). By all these and other known techniques the mRNA fractions are transferred to a recipient carrier where they are bound by an irreversible covalent bond or a strong pair bond. Consequently, these methods are only suitable for analytical purposes and cannot be used for preparative recovery of mRNA fractions, which are required for such purposes as gene therapy or genetic engineering.
In summary, it is apparent that while electrophoretic resolution of mRNA provides a good analytical method, no simple methods are known for the elution of mRNA fractions from an electrophoresis gel matrix. (See, for example, Rosen et al., (1975) Biochemistry 14, 69-78 at p. 71).
A method for the preparative recovery of individual mRNA fractions has recently been reported by Graeve et al. (1982) Biochemical and Biophysical Researach Communications 107, No. 4 1559-1565. In the introduction on page 1559 the author discusses the prior art as follows:
"Gradient centrifugation did not resolve the complex mRNA fractions very well. The method of choice is gel electrophoresis in polyacrylamide or agarose which results in an excellent resolution especialy when denaturing reagents such as formaldehyde, methylmercuric hydroxide, urea or formamide are incorporated into the gel matrix. In spite of the high resolution achieved analytically, extraction of biologically active mRNA on a preparative scale from discrete gel bands proved to be rather difficult". PA0 "The preparative separation of relatively small RNAs such as globin mRNA or snRNAs appears to be particularly attractive. Resolution of high molecular weight RNAs is less satisfactory, because one has to work under nondenaturing conditions".
The method proposed by Graeve et al. is HPLC gel filtration and the results are described by the authors on page 1564 as follows:
It is thus, seen that while there has been a long-felt need for the preparative recovery of mRNA fractions, no satisfactory method for this purpose has so far been reported. It is particularly noteworthy that in spite of such long-felt need, it never occurred to one in the scientific community to try and remove resolved mRNA fraction from an electrophoresis matrix by the reversible linkage to poly-U or poly-dT carrier sheets via H-bridges.
In accordance with the present invention, it has now surprisingly been found that the for the successful removal of mRNA fractions from an electrophoresis matrix, it is not necessary to apply irreversible covalent or strong polar bonds, and that it is also possible to apply reversible hydrogen bridge bonds such as occur upon interaction between mRNA and poly-U or poly-dT.
The present invention provides in one of its aspects a method for the preparative recovery of electrophoretically resolved mRNA fractions from the matrix of the electrophoretic resolution, comprising applying to the matrix a carrier sheet of derivative cellulose or nitrocellulose bearing a plurality of residues selected from the group of polyuridylic acid and polythymidylic acid resideus, cutting said carrier sheet into strips each of which holds an mRNA fraction and eluting an mRNA fraction from any desired strip.
Thus, in accordance with the invention there is provided a simple and effective method for the preparation of pure mRNA free of the difficulties which are inherent in known methods by which mRNA is eluted in a complex set of operations from the electrophoresis matrix itself.
The invention also provides a process for the preparative recovery of a mRNA fraction from cell material comprising extracting total RNA from the cell material by methods known per se, separating a mixture of mRNA fractions from all other RNA by means of column chromatography on derivative powderous cellulose or powderous agarose bearing a plurality of residues selected from the group of polyuridylic acid (poly-U) and poly-thymidylic acid (poly-dT) residues, isolating a total mRNA fraction, subjecting the so-isolated total mRNA fraction to electrophoresis, transferring the resolved mRNA fractions from the electrophoresis matrix to a carrier sheet of derivative cellulose or nitrocellulose bearing a plurality of residues selected from the group of poly-U and poly-dT residues, cutting said carrier sheet into strips each holding an mRNA fraction and eluting an mRNA fraction from any desired strip.
By yet another aspect, the invention provides for use in the above methods a sheet of derivative cellulose or nitrocellulose comprising a plurality of residues selected from the group of poly-U and poly-dT residues.
The invention is illustrated in the following examples without being limited thereto: